The DNA-binding domain of the gene regulatory protein AreA extends beyond the minimal zinc-finger region conserved between GATA proteins

The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91).     

Interaction of diphtheria toxin with adenylyl-(3',5')-uridine 3'-monophosphate. II. The NAD-binding site and determinants of dinucleotide affinity

Diphtheria toxin (DT) binds NAD with a KD of about 10 microM and adenylyl-(3',5')-uridine 3'-monophosphate (ApUp) with KD values ranging from 9 pM to 1.8 nM, depending on temperature (Collins, C. M., Barbieri, J. T., and Collier, R. J. (1984) J. Biol. Chem. 259, 15154-15158). Here we report experiments to explore relationships between ApUp binding and NAD binding to DT and to identify structural features of ApUp that determine its high affinity for DT. NAD, adenine, and nicotinamide competitively inhibited ApUp binding to DT, and we confirmed that ApUp blocked the binding and hydrolysis of NAD. Binding of P-site ligands to the toxin blocked interactions with ApUp. CRM197, a mutant form of DT defective in NAD binding and hydrolysis, bound ApUp 5,000-fold less tightly than did DT. These results are consistent with models in which the ApUp- and NAD-binding sites on DT overlap or are identical. Various mono-, di-, and oligonucleotides were studied as competitors of ApUp binding or the NAD-glycohydrolase reaction. The results imply that the high affinity of ApUp for DT depends on the presence of the 3'-terminal phosphate and a 3'-5' internucleoside linkage. There was strong specificity for adenine as the 5' base, but only weak specificity for uracil as the 3' base. Oligoribonucleotides containing additional nucleotides at either or both ends of ApUp sequences bound to the toxin 1-3 orders of magnitude less avidly than ApUp. Oligodeoxyribonucleotides containing dApdT sequences bound with still lower affinities. In contrast to the case with whole toxin, ApUp bound to fragment A less avidly than did NAD, and elimination of the 3'-terminal phosphate of ApUp resulted in increased affinity for the protein. These differences may reflect the absence in free fragment A of interactions with the cationic P-site, located on the toxin's B moiety.     

Structural and functional characterisation of the DNA binding domain of the Aspergillus nidulans gene regulatory protein AreA

The 876-aa protein AreA regulates the expression of numerous genes involved in nitrogen metabolism in Aspergillus nidulans, and interacts with GATA sequences upstream of the relevant genes. We have carried out limited proteolysis of the C-terminal domain of the AreA protein in order to identify possible structural domains within the protein. A stable 156-amino-acid fragment was identified that contained the zinc finger region, and this sequence was cloned and expressed in E. coli. Fluorescence spectroscopy of the purified protein showed that the proteolytic domain was folded and could be denatured by high concentrations of urea (approximately 4 M), exhibiting a sharp transition. Fluorescence spectroscopy was also used to monitor binding to a DNA duplex containing the AreA recognition site, demonstrating tight binding of the domain to its DNA recognition sequence. The DNA binding affinity of the domain is comparable with that of the native AreA protein and much higher than that of the minimal zinc finger region of AreA.     

Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362

The human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity and substrate binding cooperativity. Previous kinetic studies have suggested that Lys362 in the pigeon cytosolic NADP+-dependent malic enzyme has remarkable effects on the binding of NADP+ to the enzyme and on the catalytic power of the enzyme (Kuo, C. C., Tsai, L. C., Chin, T. Y., Chang, G.-G., and Chou, W. Y. (2000) Biochem. Biophys. Res. Commun. 270, 821-825). In this study, we investigate the important role of Gln362 in the transformation of cofactor specificity from NAD+ to NADP+ in human m-NAD-ME. Our kinetic data clearly indicate that the Q362K mutant shifted its cofactor preference from NAD+ to NADP+. The Km(NADP) and kcat(NADP) values for this mutant were reduced by 4-6-fold and increased by 5-10-fold, respectively, compared with those for the wild-type enzyme. Furthermore, up to a 2-fold reduction in Km(NADP)/Km(NAD) and elevation of kcat(NADP)/kcat(NAD) were observed for the Q362K enzyme. Mutation of Gln362 to Ala or Asn did not shift its cofactor preference. The Km(NADP)/Km(NAD) and kcat(NADP)/kcat(NAD) values for Q362A and Q362N were comparable with those for the wild-type enzyme. The DeltaG values for Q362A and Q362N with either NAD+ or NADP+ were positive, indicating that substitution of Gln with Ala or Asn at position 362 brings about unfavorable cofactor binding at the active site and thus significantly reduces the catalytic efficiency. Our data also indicate that the cooperative binding of malate became insignificant in human m-NAD-ME upon mutation of Gln362 to Lys because the sigmoidal phenomenon appearing in the wild-type enzyme was much less obvious that that in Q362K. Therefore, mutation of Gln362 to Lys in human m-NAD-ME alters its kinetic properties of cofactor preference, malate binding cooperativity, and allosteric regulation by fumarate. However, the other Gln362 mutants, Q362A and Q362N, have conserved malate binding cooperativity and NAD+ specificity. In this study, we provide clear evidence that the single mutation of Gln362 to Lys in human m-NAD-ME changes it to an NADP+-dependent enzyme, which is characteristic because it is non-allosteric, non-cooperative, and NADP+-specific.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides: ligand-induced conformational changes

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is inactivated by trypsin, chymotrypsin, pronase E, thermolysin, 4.0 M urea, and by heating to 49 degrees C. It is protected, to varying degrees, against all these forms of inactivation by glucose 6-phosphate, NAD+, and NADP+. When these ligands are present at 10 times their respective KD concentrations, protection by NAD+ or glucose 6-phosphate is substantially greater than protection by NADP+. A detailed analysis was undertaken of the protective effects of these ligands, at varying concentrations, on proteolysis of glucose-6-phosphate dehydrogenase by thermolysin. This study confirmed the above conclusion and permitted calculation of KD values for NAD+, NADP+, and glucose 6-phosphate that agree with such values determined by independent means. For NADP+, two KD values, 6.1 microM and 8.0 mM, can be derived, associated with protection against thermolysin by low and high NADP+ concentrations, respectively. The former value is in agreement with other determinations of KD and the latter value appears to represent binding of NADP+ to a second site which causes inhibition of catalysis. A Ki value of 10.5 mM for NADP+ was derived from inhibition studies. The principal conclusion from these studies is that NAD+ binding to L. mesenteroides glucose-6-phosphate dehydrogenase results in a larger global conformational change of the enzyme than does NADP+ binding. Presumably, a substantially larger proportion of the free energy of binding of NAD+, compared to NADP+, is used to alter the enzyme's conformation, as reflected in a much higher KD value. This may play an important role in enabling this dual nucleotide-specific dehydrogenase to accommodate either NAD+ or NADP+ at the same binding site.     

Binding of pyridine nucleotide coenzymes to the beta-subunit of the voltage-sensitive K+ channel

The beta-subunit of the voltage-sensitive K(+) (K(v)) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of K(v)beta2 shows NADP bound in its active site. Here we report that K(v)beta2 displays a high affinity for NADPH (K(d) = 0.1 micrometer) and NADP(+) (K(d) = 0.3 micrometer), as determined by fluorometric titrations of the recombinant protein. The K(v)beta2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K(d)(NADPH) of Q214R was 10-fold greater than the wild-type protein. The K(d)(NADPH) was unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease in K(d)(NADPH), shifted the pK(a) of K(d)(NADPH) from 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of K(v)beta2-nucleotide complexes may be formed in vivo.     

Enhancement of coenzyme binding by a single point mutation at the coenzyme binding domain of E. coli lactaldehyde dehydrogenase

Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) share some structural and kinetic properties. One difference is that PAD can use NAD+ and NADP+, whereas ALD only uses NAD+. An acidic residue has been involved in the exclusion of NADP+ from the active site in pyridine nucleotide-dependent dehydrogenases. However, other factors may participate in NADP+ exclusion. In the present work, analysis of the sequence of the region involved in coenzyme binding showed that residue F180 of ALD might participate in coenzyme specificity. Interestingly, F180T mutation rendered an enzyme (ALD-F180T) with the ability to use NADP+. This enzyme showed an activity of 0.87 micromol/(min * mg) and K(m) for NADP+ of 78 microM. Furthermore, ALD-F180T exhibited a 16-fold increase in the V(m) /K(m) ratio with NAD+ as the coenzyme, from 12.8 to 211. This increase in catalytic efficiency was due to a diminution in K(m) for NAD+ from 47 to 7 microM and a higher V(m) from 0.51 to 1.48 micromol/(min * mg). In addition, an increased K(d) for NADH from 175 (wild-type) to 460 microM (mutant) indicates a faster product release and possibly a change in the rate-limiting step. For wild-type ALD it is described that the rate-limiting step is shared between deacylation and coenzyme dissociation. In contrast, in the present report the rate-limiting step in ALD-F180T was determined to be exclusively deacylation. In conclusion, residue F180 participates in the exclusion of NADP+ from the coenzyme binding site and disturbs the binding of NAD+.     

The 2'-phosphate of NADP is responsible for proper orientation of the nicotinamide ring in the oxidative decarboxylation reaction catalyzed by sheep liver 6-phosphogluconate dehydrogenase

Sheep liver 6-phosphogluconate dehydrogenase shows a high specificity for NADP, with a much lower affinity for NAD. Discrimination between NADP and NAD suggests that the interactions between the 2'-phosphate and 6-phosphogluconate dehydrogenase contribute most of the binding energy for NADP. There are three active site residues, Asn-32, Arg-33, and Thr-34, that hydrogen-bond to the 2'-phosphate of NADP according to the crystal structure of the E.Nbr(8)ADP complex. In this study alanine mutagenesis was used to probe the contribution of each of the three residues to binding the cofactor and to catalysis. All mutant enzymes exhibit no significant change in V/E(t) or K(6PG) but an increase in K(NADP) that ranges from 6- to 80-fold. All mutant enzymes also exhibit at least a 7-fold increase in the primary kinetic (13)C-isotope effect-1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Asn-32, Arg-33, and Thr-34 in providing binding energy for NADP, and more importantly, the 2'-phosphate of NADP is required for proper orientation of the cofactor to allow rotation about the N-glycosidic bond as it is reduced in the hydride transfer step.     

Probing the affinity and specificity of yeast alcohol dehydrogenase I for coenzymes.

Yeast (Saccharomyces cerevisiae) alcohol dehydrogenase I (SceADH) binds NAD+ and NADH less tightly and turns over substrates more rapidly than does horse (Equus caballus) liver alcohol dehydrogenase E isoenzyme (EcaADH), and neither enzyme uses NADP efficiently. Amino acid residues in the proposed adenylate binding pocket of SceADH were substituted in attempts to improve affinity for coenzymes or reactivity with NADP. Substitutions in SceADH (Gly202Ile or Ser246Ile) with the corresponding residues in the adenine binding site of the homologous EcaADH have modest effects on coenzyme binding and other kinetic constants, but the Ser246Ile substitution decreases turnover numbers by 350-fold. The Ser176Phe substitution (also near adenine site) significantly decreases affinity for coenzymes and turnover numbers. In the consensus nucleotide-binding betaalphabeta fold sequence, SceADH has two alanine residues (177-GAAGGLG-183) instead of the Leu200 in EcaADH (199-GLGGVG-204); the Ala178-Ala179 to Leu substitution significantly decreases affinity for coenzymes and turnover numbers. Some NADP-dependent enzymes have an Ala corresponding to Gly183 in SceADH; the Gly183Ala substitution significantly decreases affinity for coenzymes and turnover numbers. NADP-dependent enzymes usually have a neutral residue instead of the Asp (Asp201 in SceADH) that interacts with the hydroxyl groups of the adenosine ribose, along with a basic residue (at position 202 or 203) to stabilize the 2'-phosphate of NADP. The Gly203Arg change in SceADH does not significantly affect the kinetics. The Gly183Ala or Gly203Arg substitutions do not enable SceADH to use NADP+ as coenzyme. SceADH with the single Asp201Gly or double Asp201Gly:Gly203Arg substitutions have similar, low activity with NADP+. The results suggest that several of the amino acid residues participate in coenzyme binding and that conversion of specificity for coenzyme requires multiple substitutions.     

Complete reversal of coenzyme specificity by concerted mutation of three consecutive residues in alcohol dehydrogenase

Gastric tissues from amphibian Rana perezi express the only vertebrate alcohol dehydrogenase (ADH8) that is specific for NADP(H) instead of NAD(H). In the crystallographic ADH8-NADP+ complex, a binding pocket for the extra phosphate group of coenzyme is formed by ADH8-specific residues Gly223-Thr224-His225, and the highly conserved Leu200 and Lys228. To investigate the minimal structural determinants for coenzyme specificity, several ADH8 mutants involving residues 223 to 225 were engineered and kinetically characterized. Computer-assisted modeling of the docked coenzymes was also performed with the mutant enzymes and compared with the wild-type crystallographic binary complex. The G223D mutant, having a negative charge in the phosphate-binding site, still preferred NADP(H) over NAD(H), as did the T224I and H225N mutants. Catalytic efficiency with NADP(H) dropped dramatically in the double mutants, G223D/T224I and T224I/H225N, and in the triple mutant, G223D/T224I/H225N (kcat/KmNADPH = 760 mm-1 min-1), as compared with the wild-type enzyme (kcat/KmNADPH = 133330 mm-1 min-1). This was associated with a lower binding affinity for NADP+ and a change in the rate-limiting step. Conversely, in the triple mutant, catalytic efficiency with NAD(H) increased, reaching values (kcat/KmNADH = 155000 mm-1 min-1) similar to those of the wild-type enzyme with NADP(H). The complete reversal of ADH8 coenzyme specificity was therefore attained by the substitution of only three consecutive residues in the phosphate-binding site, an unprecedented achievement within the ADH family.     

Fragment reconstitution of a small protein: disulfide mutant of a short C-terminal fragment derived from streptococcal protein G

Hierarchical studies on the folding of protein G B1 domain have shown that the C-terminal fragment (C16) has a considerable amount of beta-hairpin structure that exchanges between the folded and unfolded states at room temperature, and that the C16 fragment binds noncovalently to an N-terminal fragment (N40) under physiological conditions. Those studies have led us to the hypothesis that the amphipathic beta-hairpin structure of C16 initiates folding of the domain. To obtain a more detailed understanding of the folding mechanism of the domain, we designed a mutant of C16 (SS16ox) with a disulfide bond between residues 41 and 56, and then examined the interaction of the mutant with N40 by surface plasmon resonance (SPR) and by thermal denaturation studies using circular dichroism. SS16ox strongly interacted with N40, with an equilibrium constant, KD, that was 7-fold higher than wild-type. The association rate constant, kon, of SS16ox was 8.7-fold higher than that of wild-type. This strong interaction can be explained by the entropic effect of the disulfide bond. The introduction of the disulfide bond into C16 stabilizes the beta-hairpin structure of C16, accelerates the association rate with N40, and then stabilizes the whole complex. These results support a hypothetical folding mechanism of protein G where the amphipathic beta-hairpin structure of C16 acts as a nucleus and accelerates folding of the whole molecule.     

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.     

The role of invariant amino acid residues at the hydride transfer site of proton-translocating transhydrogenase

Transhydrogenase couples proton translocation across a membrane to hydride transfer between NADH and NADP+. Previous x-ray structures of complexes of the nucleotide-binding components of transhydrogenase ("dI2dIII1" complexes) indicate that the dihydronicotinamide ring of NADH can move from a distal position relative to the nicotinamide ring of NADP+ to a proximal position. The movement might be responsible for gating hydride transfer during proton translocation. We have mutated three invariant amino acids, Arg-127, Asp-135, and Ser-138, in the NAD(H)-binding site of Rhodospirillum rubrum transhydrogenase. In each mutant, turnover by the intact enzyme is strongly inhibited. Stopped-flow experiments using dI2dIII1 complexes show that inhibition results from a block in the steps associated with hydride transfer. Mutation of Asp-135 and Ser-138 had no effect on the binding affinity of either NAD+ or NADH, but mutation of Arg-127 led to much weaker binding of NADH and slightly weaker binding of NAD+. X-ray structures of dI2dIII1 complexes carrying the mutations showed that their effects were restricted to the locality of the bound NAD(H). The results are consistent with the suggestion that in wild-type protein movement of the Arg-127 side chain, and its hydrogen bonding to Asp-135 and Ser-138, stabilizes the dihydronicotinamide of NADH in the proximal position for hydride transfer.     

Critical residues for the coenzyme specificity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biological inactivation of prostaglandins. Sequence alignment within SDR coupled with molecular modeling analysis has suggested that Gln-15, Asp-36, and Trp-37 of 15-PGDH may determine the coenzyme specificity of this enzyme. Site-directed mutagenesis was used to examine the important roles of these residues. Several single mutants (Q15K, Q15R, W37K, and W37R), double mutants (Q15K-W37K, Q15K-W37R, Q15R-W37K, and Q15R-W37R), and triple mutants (Q15K-D36A-W37R and Q15K-D36S-W37R) were prepared and expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli and purified by GSH-agarose affinity chromatography. Mutants Q15K, Q15R, W37K, W37R, Q15K-W37K, and Q15R-W37K were found to be inactive or almost inactive with NADP+ but still retained substantial activity with NAD+. Mutant Q15K-W37R and mutant Q15R-W37R showed comparable activity for NAD+ and NADP+ with an increase in activity nearly 3-fold over that of the wild type. However, approximately 30-fold higher in K(m) for NADP+ than that of the wild type enzyme for NAD+ was found for mutants Q15K-W37R and Q15R-W37R. Similarly, the K(m) values for PGE(2) of mutants were also shown to increase over that of the wild type. Further mutation of Asp-36 to either an alanine or a serine of the double mutant Q15K-W37R (i.e., triple mutants Q15K-D36A-W37R and Q15K-D36S-W37R) rendered the mutants exhibiting exclusive activity with NADP+ but not with NAD+. The triple mutants showed a decrease in K(m) for NADP+ but an increase in K(m) for PGE(2). Further mutation at Ala-14 to a serine of a triple mutant (Q15K-D36S-W37R) decreased the K(m) values for both NADP+ and PGE(2) to levels comparable to those of the wild type. These results indicate that the coenzyme specificity of 15-PGDH can be altered from NAD+ to NADP+ by changing a few critical residues near the N-terminal end.     

The heterotrimer of the membrane-peripheral components of transhydrogenase and the alternating-site mechanism of proton translocation

Transhydrogenase undergoes conformational changes to couple the redox reaction between NAD(H) and NADP(H) to proton translocation across a membrane. The protein comprises three components: dI, which binds NAD(H); dIII, which binds NADP(H); and dII, which spans the membrane. Experiments using isothermal titration calorimetry, analytical ultracentrifugation, and small angle x-ray scattering show that, as in the crystalline state, a mixture of recombinant dI and dIII from Rhodospirillum rubrum transhydrogenase readily forms a dI(2)dIII(1) heterotrimer in solution, but we could find no evidence for the formation of a dI(2)dIII(2) tetramer using these techniques. The asymmetry of the complex suggests that there is an alternation of conformations at the nucleotide-binding sites during proton translocation by the complete enzyme. The characteristics of nucleotide interaction with the isolated dI and dIII components and with the dI(2)dIII(1) heterotrimer were investigated. (a) The rate of release of NADP(+) from dIII was decreased 5-fold when the component was incorporated into the heterotrimer. (b) The binding affinity of one of the two nucleotide-binding sites for NADH on the dI dimer was decreased about 17-fold in the dI(2)dIII(1) complex; the other binding site was unaffected. These observations lend strong support to the alternating-site mechanism.